Amongst insect borne viral diseases, Japanese encephalitis and dengue have a notoriety of encompassing entire region of South East Asia. The envelope glycoprotein contains at least five determinants that seem to be correlated with the important biological properties of hemagglutination and neutralization. Envelope glycoprotein is responsible for the attachment of the virus and thus is associated with infectivity. The existing vaccine for Japanese encephalitis is purified, killed virus vaccine prepared from infant mouse brain that consists of mainly envelope glycoprotein of the virus. Three injections of mouse brain derived killed purified vaccine followed by a booster after 12 to 18 months, can give effective immunization as judged by induction of neutralizing antibodies. Mouse brain vaccine shows neutralizing antibodies against Indian strain also. The efficacy of the vaccine has been noted only if three doses are administered. U.S. Pat. No. 5,824,506 Chan, et al. Oct. 20, 1998 discloses peptide antigens derived from the dengue virus type-2 glycoprotein NS1. The peptide antigens are specifically immunoreactive with sera from individuals infected with the dengue virus. The antigens are useful as diagnostic tools in determining whether an individual has been or is infected with dengue virus, and for discriminating between infection with dengue virus and infection with related flaviviruses. The antigens are also useful in vaccine compositions for immunizing individuals against infection with the dengue virus.
U.S. Pat. No. 5,494,671 Lai, et al. discloses C-terminally truncated flavivirus envelope proteins 80-81% in size which are more immunogenic than their counterpart full-length proteins. The aforesaid patent further discloses recombinant viruses that encode the truncated protein and to host cells infected therewith. Host cells express the truncated protein on their outer membrane and secrete it into the medium. The patent discloses vaccines for use against flavivirus infection. The vaccines include either a recombinant vaccinia virus expressing the truncated envelope protein, and the truncated envelope protein produced by a recombinant baculovirus.
In China, attenuated Japanese encephalitis virus vaccine consisting of Japanese encephalitis virus strain SA 14-14-2 is being used. The attenuation has been carried out in hamster kidney cells and have unknown passage histories. Mice inoculated intracerebrally with the SA 14-14-2 vaccine strains survived without showing any signs of CNS involvement. The virus titers in brains persisted at low levels for several days and could not be detected after 10 days (Hase T, Dubois, Dr. Summers, P L, Downs, M B and Ussery, M A. (1993) Comparison of replication rates and pathogenicities between the SA14 parent and SA14-14-2 vaccine strains of Japanese encephalitis virus in mouse brain neurons. Arch Virol 130 131-43. Dubois T., Dr. Summers, P L. Downs, M B and Ussery, M A (1993) Comparison of replication rates and pathogenicities between the SA14 parent and SA 14-14-2 vaccine strains of Japanese encephalitis virus in mouse brain neurons. Arch Virol 130 131-43). The safety and immunogenicity of this vaccine has been tested. 1,026 children between the ages of 5 and 12 years, were vaccinated with live-attenuated Japanese encephalitis virus vaccine. None of the group of 47 of the vaccinated children, has temperature >37.4° C. Seroconversion rates in seronegative children were 100% (GMT 35.3) (Xin Y Y, Ming, Z G, Peng, G Y, Jain, A and Min, L H (1988) Safety of a live-attenuated Japanese encephalitis virus vaccine (SA14-14-2) for children. Am J Trop Med Hyg 39 214-7). Many attempts are being carried out to develop recombinant vaccine for Japanese encephalitis virus. These include expression of various proteins and then immunizing animals with the products. It was realized very early that expression and immunization with envelope glycoprotein alone was not very useful (Mason P W, McAda, P C, Dalrynple, J M, Fournier, M J and Mason, T L (1987) Expression of Japanese encephalitis virus antigens in Escherichia coli. Virology 158 361-72). Mice immunized with recombinant baculovirus infected cells containing envelope glycoprotein and NS-1 genes were challenged with Japanese encephalitis virus. Survival was increased from about 30% in unimmunized mice to 70% in envelope glycoprotein and polyprotein recipients but not in NS-1 recipients (McCown J, Cochran, M, Putnak, R, Feighny, R, Burrous, J, Henchal, E and Hoke, C (1990). Protection of mice against lethal Japanese encephalitis with a recombinant baculovirus vaccine. Am J Trop Med Hyg 42 491-9). Immunization of mice with purified extracellular subviral particles composed of prM and E proteins in recombinant vaccinia viruses could protect mice from 4.9×105 LD50 of Japanese encephalitis virus. (Konishi E, Pincus, S, Paoletti, E, Shope, R E, Burrage, T and Mason, P W. (1992) Mice immunized with a subviral particle containing the Japanese encephalitis virus prM/M and E proteins are protected from lethal JEV infection. Virology 188 714-20). These particulate antigens were also shown to induce Japanese encephalitis virus specific CTL response in mice. (Konishi E, Win, K S, Kurane, I, Mason, P M, Shope, R E and Ennis, F A (1997) Particulate vaccine candidate for Japanese encephalitis induces long-lasting virus-specific memory T lymphocytes in mice. Vaccine 15 281-6).
Vaccinia recombinants that co-expressed the genes for premembrane and envelope glycoprotein elicited high levels of neutralizing and HI antibodies in mice and protected mice from a lethal challenge by Japanese encephalitis virus. Recombinants expressing only the gene for NS1 induced antibodies to NS1 but provided low levels of protection from a similar challenge dose of Japanese encephalitis virus. Immunization of mice with vaccinia recombinant viruses containing PrM gene along with NS-1 and envelope glycoprotein protected them from challenge with Japanese encephalitis virus. Pox virus (Canary pox and vaccinia) based Japanese encephalitis recombinant vaccines have been constructed and shown to produce Japanese encephalitis virus specific CTLs in mice. (Konishi, E, Kurane, I Mason, P W, Shope, R E and Ennis, F A (1997) Poxvirus-based Japanese encephalitis vaccine candidates induce J E virus specific CD8+ cytotoxic T lymphocytes in mice. Virology 227 353). Poxvirus-based recombinant J E vaccine candidates, NYVAC-JEV and ALVAC-JEV, encoding the Japanese encephalitis virus prM, E and NS1 proteins were examined for their ability to induce Japanese encephalitis virus-specific CTLs. The volunteers received subcutaneous inoculations with each of these candidates on days 0 and 28. Anti-E and anti-NS1 antibodies were elicited in a most vaccinees inoculated with NYVAC-JE virus and in some vaccinees inoculated with ALVAC-JEV, PBMCs obtained from approximately one half of vaccinees showed positive proliferation in response to stimulation with live Japanese encephalitis virus. Presence of the Japanese encephalitis virus-specific CDS+CD4− cytotoxic T cells in vitro-stimulated peripheral blood mononuclear cells obtained from two NYVAC-JEV and two ALVAC-JEV vaccinees was demonstrated. (Konishi E, Kurane, I, Mason, P M, Shope, R E, Kanesa-Thasan, N, Smucny, J J, Hoke, C H Jr and Ennis, F A (1998). Induction of Japanese encephalitis virus-specific cytotoxic T lymphocytes in humans by poxvirus-based J E vaccine candidates. Vaccine 16 842-9). A chimeric Yellow fever (YF)/JE virus (ChimeriVax-JE virus) was constructed by insertion of the prM and envelope glycoprotein genes of an attenuated human vaccine strain (SA14-14-2) of Japanese encephalitis virus between C and NS genes of a YF 17D infectious clone. Mice inoculated subcutaneously with one dose of >/=103 pfu of ChimeriVax-JE virus were protected against IP challenge with a virulent Japanese encephalitis virus.
In recent years, it has been shown that fragments of proteins in the form of synthetic peptides can be used to induce T helper and antibody responses. Attempts to delineate B cell epitopes from Japanese encephalitis virus have resulted in delineation of Met 303 to Trp 396 as the shortest sequence capable of reacting with 10 Mabs. Disulfide bond between cys 304 and 335 was required for presentation of the binding site(s) for these Mabs. However, it was not an effective immunogen for inducing neutralizing or protective antibodies in mice (Mason P W, Dalrymple, J M, Gentry, M K, McCown, J M, Hoke, C H, Burke, D S, Fournier, M J and Mason, T L (1989) Molecular characterization of a neutralizing domain of the Japanese encephalitis virus structural glycoprotein. J Gen Virol 70 2037-49). The fragment carrying the coding sequence of amino acid 373-399 of envelope glycoprotein elicited the highest neutralizing antibody titer (1:75). HI antibodies were not induced by this fusion protein (Seif S A, Korita, K and Igarashi, A (1996) A 27 amino acid coding region of E virus protein expressed in E. coli as fusion protein with glutathione-S-transferase elicit neutralizing antibody in mice. Virus Res 43 91-6). Neutralizing antibody inducing epitopes have been detected on C terminal regions of envelope glycoprotein (Seif S A, Morita, K, Matsuo, S, Hasebe, F and Igarashi, A (1995) Finer mapping of neutralizing epitope(s) on the C-terminal of Japanese encephalitis virus E-protein expressed in recombinant Escherichia coli system. Vaccine 13 1515-21 and Jan L R, Yang, C S, Henchal, L S, Sumiyoshi, H, Summers, P L, Dubois D R and Lai, C J (1993) Increased immunogenicity and protective efficacy in outbred and inbred mice by strategic carboxyl-terminal truncation of Japanese encephalitis virus envelope glycoprotein. Am J Trop Med Hyg 48 412-23) Peptides from C protein have also been delineated for reactivity with sera from Japanese encephalitis and dengue patients. Pep91-105 and 8-22 belonged to group-specific epitopes that reacted with both Japanese encephalitis and dengue-1 patient sera. Pep 1-15 and 34-48 belonged to subcomplex-specific epitopes that reacted only with Japanese encephalitis but not with dengue-1 patient sera. (Huang J H, Wey, J J, Lee, H F, Tsou) T L, Wu, C S, Wu, J R, Chen, H M, Chin, C, Chien, L J, Chen, L K, Wu Y C, Pan, M J and Wang, T M (1996) Identification of immunodominant, group-specific and subcomplex-specific, continuous epitopes in the core regions of Japanese encephalitis virus using synthetic peptides. Virus Res 41 43-53).